Changes in the rates of protein synthesis and degradation in the tail of Rana catesbeiana tadpoles during normal metamorphosis

DEVELOPMENTAL

Changes

BIOLOGY

30, 366-373

(1973)

in the Rates of Protein

Synthesis

in the Tail of Rana catesbeiana

Tadpoles

and Degradation During

Normal

Metamorphosis’ G. H. Department

of Chemistry,

LITTLE’,

Florida

State

B. G.

ATKINSON~,

University, Accepted

Tallahassee, August

AND Florida

32,306

in the tadpole tail during developed which permits dethe rate of protein synthesis. followed later by an increase results to the mechanism of

Increases in the rate of protein degradation have not been directly observed previously in either spontaneous or induced metamorphosis, but have been inferred from increases in the activity of proteolytic enzymes. The effect of triiodothyronine (T,) on the rate of protein synthesis in the tadpole tail has been studied in vitro by Tata (1966) and in viuo by Tonoue and Frieden (1970). The former investigator observed an increase in the rate of protein synthesis in response to T,; Tonoue and Frieden observed a decrease. Further investigation of this problem is obviously necessary. Before undertaking such studies of induced metamorphosis, however, experiments should first be carried out on animals undergoing spontaneous metamorphosis so that results obtained in response to thyroid hormones can be interpreted in terms of the events of normal development. For this reason the present investigation was undertaken. In order to establish the time sequence of alterations in the balance between the rates of protein synthesis and degradation during metamorphic resorption of the tadpole tail, we developed a new dual labeling procedure which permitted us to determine the rate of protein degradation independently of the rate of protein

INTRODUCTION

Protein, which comprises roughly onefourth of the tissue dry weight, is the major cell constituent to be disposed of during the metamorphic regression of the tadpole tail. A net loss of protein could be achieved by a decrease in the rate of protein synthesis, an increase in the rate of protein degradation or both. Investigators in the field have concentrated primarily on studies of the increases which occur in the activities of several proteolytic enzymes during metamorphosis (see reviews by Atkinson, 1971; Frieden and Just, 1970; Weber, 1967). While these findings are significant, a more detailed knowledge of the balance between the rates of protein synthesis and degradation should contribute to our understanding of the mechanism of tail resorption. ‘This research was supported by U.S. Public Health Service Grant HD 01236 from the National Institute of Child Health and Human Development. This is paper No. 49 in a series from this laboratory on the biochemistry of amphibian metamorphosis. *Present address: Department of Biochemistry, Texas Technological University School of Medicine, P.O. Box 4569, Lubbock, Texas 79409. a National Institutes of Health Postdoctoral Fellow, No. HD41,746. Present address: Department of Zoology, The University of Western Ontario, London 72, Ontario. 366 0 1973 by Academic Press, Inc. of remoduction in anv form reserved.

FRIEDEN

2, 1972

The rates of protein synthesis and degradation were investigated metamorphosis. A new dual radioisotope labeling procedure was termination of the rate of protein degradation independently of An early depression of protein synthesis was observed which was in the rate of protein degradation. The relationship of these tail resorption is discussed.

Copyright All rinhts

E.

LITTLE,

ATKINSON,

AND

FRIEDEN

Protein Tbmover

synthesis during the course of normal metamorphosis. This procedure involved prelabeling the protein with 3H-leucine for 5 days and then pulse labeling with 14C-leucine. The rate of increase of the 3H: l*C ratio in the free amino acid pool provided a measure of the rate of protein degradation. A separate study of changes in the rate of protein synthesis was carried out using the rate of incorporation of a ‘%-labeled amino acid mixture into protein as a measure of the rate of protein synthesis. METHODS

AND

MATERIALS

Radioisotopes. All labeled compounds used in these studies were purchased from New England Nuclear. Radioactive samples were counted with a Beckman Model LS-250 liquid scintillation spectrometer in a cocktail consisting of 0.5% PPO, 10% naphthalene, and 4% Cab-OSil in dioxane. Calculations of dpm were performed by means of computer programs as described by Eaton (1971). Animals. Rana catesbeiana tadpoles were obtained from a commercial supplier, and maintained on a diet of collard greens. The tadpole water was changed daily. Tadpoles were staged according to criteria established by Taylor and Kollros (1946). Treatment of tadpoles. All experiments were performed during late spring and early summer (1971). For the study of protein synthesis, tadpoles were chosen at specific stages of metamorphosis and injected intramuscularly in the back with a commercial 14C amino acid mixture (200 Ci/mm, NEN Cat. No. NEN 445) at a dose of 0.2 &i/g body weight. After 1-hr labeling, the tadpoles were individually anesthetized with 0.1% solution of TriCaine methane sulfonate (K & K Laboratories, Inc.) and thoroughly bled from the truncus arteriosus. Tails were completely excised, weighed, and either used di-

during

Metamorphosis

367

rectly or frozen on Dry Ice and stored at - 20°C before homogenization. In the protein degradation experiment 36 preclimax tadpoles were given an initial intraperitoneal injection of 1.0 &X/g body weight of 3H-leucine which was followed on alternate days by additional injections of 0.5 &i/g body weight for the duration of the experiment. Beginning 5 days after the initial injection of 3H-leucine, 9 animals, chosen at random every third day for sacrifice, were each given an intraperitoneal injection of 0.5 &i/g body weight of 14C-leucine and divided into three groups of 3 tadpoles each. The first group was sacrificed after 30 min, the second group after 60 min and the third group after 180 min. After anesthetization and bleeding as described above, the terminal 3-cm portion of the tail was excised and immediately frozen on a block of Dry Ice and stored at -20°C until processed. The metamorphic stage of each animal was determined at the time of sacrifice. The experiment was immediately repeated on a second group of 36 tadpoles and the results combined with those from the first group of animals. Homogenization

of extraction

of tissue.

The procedure described for processiqs; the tail tissue follows the lipid extraction procedure described by Folch et al. (1957). The tails were homogenized in distilled water with a Teflon-glass homogenizer and diluted to a final volume of 2 ml/100 mg of tissue. A 0.5 ml aliquot of homogenate was immediately added to 9.5 ml of cold 2: 1 chlorofrom-methanol in a 12 ml, graduated, screw-capped centrifuge tube. Two milliliters of deionized H,O were then added, and the mixture was shaken thoroughly and spun in an International clinical centrifuge to separate the phases. A three-phase system resulted, with an upper aqueous phase, a lower organic phase, and a precipitate at the interface containing the macromolecules, Unincorporated labeled leucine is completely extracted into the upper phase and pro-

368

DEVELOPMENTAL

BIOLOGY

tein is completely precipitated by this procedure. The upper phase was removed and placed into a 12 ml graduated centrifuge tube. The interface was washed twice with fresh portions of pure upper phase (Folch et al., 1957), and the washings were added to the original upper phase. The combined material was diluted to 10 ml with pure upper phase, and an aliquot was taken for amino acid determination by the method of Yemm and Cocking (1955) using L-alanine as the standard. The remainder was treated with activated charcoal to remove nucleotides and centrifuged as before. The supernatant was transferred to another centrifuge tube, and about 1 ml of Dowex 50 X12 in the potassium form was added to bind the amino acids. This was allowed to equilibrate with shaking for 1 hr and then centrifuged. The supernatant was discarded and 1.0 ml of 1.0 M potassium hydroxide was added to the resin to extract the amino acids. This was allowed to stand for an hour and then centrifuged. The 3H: “C ratio was determined by liquid scintillation counting of an aliquot of the supernatant. After removal of the upper phase of the chloroform-methanol-water extract as described above, methanol was added to

CHANGES Tail wt km)

v X xv XVII XVIII XIX xx XXI XXII XXIII XXIV

1.82 2.11 2.22 2.37 2.50 2.61 2.56 1.81 1.09 0.32 0.16

IN TAIL

ANATOMICAL

Length0 (mm)

42.0 56.1 71.9 71.8 76.8 73.7 70.1 50.9 19.6 6.4 1.7

* * * * zt A * * i * *

1.3 1.1 1.9 1.3 2.6 1.6 1.1 2.1 2.0 0.6 0.3

VOLUME

30, 1973

the remaining lower phase and precipitate to give a final volume of 12 ml. This lowers the specific gravity of the lower phase, allowing the precipitate the settle. The samples were mixed and centrifuged, the supernatant was discarded. The precipitate was washed once with acetone to remove residual solvents and soluble material, and then suspended in 1 ml of 0.5 M perchloric acid and heated at 70°C for 15 min to hydrolyze nucleic acids. The mixture was then centrifuged, and the supernatant was discarded. The precipitate which contained the protein was dissolved in 1.0 M NaOH and the protein was determined by the method of Lowry et uf. (1951). The levels of 3H and 14C in the protein were determined by liquid scintillation counting. Incubation of homogenates in vitro. Duplicate O.l-ml aliquots of the original homogenate were added to 0.1 ml of 0.5 M formate buffer (pH 3.3) and to 0.1 ml of 0.5 M phosphate buffer (pH 6.8) in 10 x 75 mm test tubes in an ice bath. The mixtures were incubated at 25°C for 0, 30, and 60 min. The reaction was stopped by placing the tubes in an ice bath for 5 min. This was followed by the addition of 1.0 ml of 3% trichloroacetic acid and 0.1 ml of unlabeled 30% tail homogenate as a carrier.

TABLE 1 AND BIOCHEMICAL PARAMETERS

DURING METAMORPHOSIS

m g Protein g m tlSS”e

1.68 1.86 1.89 1.84 1.86 1.84 1.85 1.39 0.52 0.18 0.05

D Taylor and Kollros (1946). b Measured from cloaca1 opening c From different group of animals

i zt i * * + * * * + *

0.05 0.03 0.04 0.03 0.04 0.04 0.02 0.07 0.06 0.02 0.01

42.3 37.9 32.2 34.3 33.5 33.2 31.5 44.6 59.9 53.7

zt zt k * + + f i * *

2.1 2.6 1.5 1.4 2.5 1.9 1.4 2.7 5.4 4.8

-

to tip of tail. than other data

in table.

1.37 1.37 1.36 1.32 1.39 1.36 1.47 2.10 3.12 4.69

* 0.20 * 0.13 + 0.13 * 0.06 + 0.25 + 0.15 * 0.17 ztz 0.32 zt 0.24 zt 0.31

-

30.9 27.7 23.7 25.9 24.1 24.4 21.4 21.2 19.2 11.4 -

WI Amino acid/mg tissueC x 10”

em Amino aadlmg protein’ x 10’

-

-

7.2 8.9 10.5 10.6 16.8

-

+ * i + +

0.5 0.5 0.8 0.4 1.7

1.33 1.50 1.27 1.28 1.41

-

* i + f *

0.07 0.16 0.09 0.05 0.07

LIWLF,

ATKINSON,

AND

FRIEDEN

Protein

The tubes were then centrifuged for 20 min at 3000 g, and the 3H: 14C ratio was determined in aliquots of the supernatant by means of liquid scintillation counting. Incorporation of 3H-thymidine into tadpole tail DNA. Changes in the rate of incorporation of 3H-thymidine into tadpole tail DNA were investigated with the experimental procedures described by Atkinson et al. (1972).

Turnover

during

369

Metamorphosis

‘“,LLLL;” DAYS

CT

BETWEEN

STAGES-

1.4 3.3 2.9 2.5 1.5

1.6-

-0.8

P 1.2‘z B

-0.6

0 .x .E 2 B

-s 7 f e P d E f B

RESULTS

The first grossly observable signs of tail regression appear between stages XX and XXI, during which time the tail loses weight and a decrease occurs in the tail length as well as the tail length: body length ratio (Table 1). These changes continue progressively until stage XXV, when the tail is completely resorbed. During the same period the quantity of DNA, protein and free amino acids per unit weight of tissue increases (Table 1). The quantity of free amino acids per milligram of protein remains unchanged. A decline in the protein :DNA ratio begins between stages XIX and XX. Figure 1 shows the rates of incorporation of a 14C-labeled amino acid mixture into the tail protein of stages V through XXIII. The rate began to decrease between stages XVII and XVIII and decreased steadily until stage XXI, then leveled off between stages XXI and XXII (Fig. 1, solid line). The dashed line of Fig. 1 shows the changes in the radioactivity of the free amino acid pool during metamorphosis expressed as dpm/mg protein. At those stages where the rate of incorporation into protein decreases, the radioactivity of the amino acid pool increases. Since there is no change in the quantitiy of free amino acids per milligram of protein (Table l), the increase in the radioactivity of the amino acid pool (Fig. 1, dashed line) also represents an increase in pool specific activity. The changes in the rate of protein degradation which occur in vivo during

v k 0.0’ 3 4

’

X

x+’

XVI STAGES OF

xi/II

i 10.0

’ XIX ’ xk ’ xdlll XVIII xx XXII XXIV METAMORPHOSIS

FIG. 1. The in vivo rate amino acid mixture into the

of incorporation of “C TCA-precipitahle fractions (O--O) and the TCA-soluble fractions (x--x) of tadpole tail at various stages of spontaneous metamorphosis. All animals were labeled with W-labeled amino acids 60 min before sacrifice. Each point represents the mean of six separate experiments, the vertical bars signify the standard error of the mean. Notation and values in upper portion of figure entitled “Days between Stages” represents the mean, obtained from 12 tadpoles, of the time required to complete the transition from one stage to the next stage. The transition from stage X to stage XIX normally occurs in 6-12 months.

metamorphosis are shown in Fig. 12. A dramatic increase in the rate of degradation occurred between stages XX and XXI. A further increase occurred between stages XXI and XXII. Figure 2 also shows the rate of incorporation of 14C-leucine into protein during the degradation experiment. These data are in agreement those presented in Fig. 1, and illustrate the reciprocal relationship which exists between incorporation of amino acids into protein and protein degradation. Investigation of the rates of protein degradation in vitro at pH 3.3 (Fig. 3) revealed the same general trend as observed in vivo although the largest difference in the rate of

370

DEVELOPMENTAL

BIOLOGY

VOLUME

32

-2’z(D

30,

1973

c

-18 %

-15

2 2. = F

-122 ,” -. -9

s 15

-6

g’ x

-3

& z

s b

24 24

r. STAGES

OF

METAMORPHOSIS

FIG. 2. The in uiuo rate of tadpole tail protein degradation and incorporation of “C-leucine into protein at various stages of metamorphosis. The crosshatched bars represent the rates of protein degradation obtained by our dual isotope procedure and expressed as mg protein degraded per minute per mg of tissue for each stage. Time points of 30-min, 1-hr, and 3-hr were obtained for each stage with 3-6 animals per point. The open bars represent the slopes of the lines obtained when protein “C specific activity was plotted versus time for each stage. Time points of 30 and 60 min were obtained for each stage, with 3-6 animals per point.

degradation shifted from stages XX-XXI to XXI-XXII. At pH 6.8 the rates of protein degradation in vitro were too low to be measured reliably. Figure 4 shows the rate of incorporation of 3H-thymidine into DNA during metamorphosis. These results indicate that the rate of DNA synthesis in the tadpole tail begins to decrease between stages XIX and XX and continues to decrease until the tail is completely resorbed. DISCUSSION

Most prior studies of protein degradation have been carried out by following the decay of protein specific activity with time after a single injection of a radioactive amino acid. The most elegant of

XVIII

XIX STAGES

xx OF

XXI

METAMORPHOSIS

FIG. 3. The in vitro rate of tadpole tail protein degradation at various stages of metamorphosis. The bars represent the rates of protein degradation obtained by our dual isotope procedure and expressed as mg protein degraded per minute per mg of tissue for each stage. Tail homogenates were incubated at 25°C in pH 3.3 formate buffer (0.5 A4), for 0, 30, and 60 min.

these experiments was described by Arias et al. (1969). These investigators employed two isotopic forms of the same amino acid injected 4-6 days apart so that two time points could be obtained from the same animal. A disadvantage of methods based on the rate of decay of protein specific activity is that the rate of decay is dependent upon the rate of protein synthesis as well as the rate of degradation. As Arias et al. (1969) pointed out, their method is based upon the assumption that the rate of protein synthesis is the same at the time of both isotope administrations. Since the rate of protein synthesis undergoes marked changes in the tadpole tail during metamorphosis it was necessary for us to devise a method for determining the rate of protein degradation independently of the rate of protein synthesis. The most obvious method would

LITTLE ATKINSON,

AND FRIEDEN

Protein

5.0~2.5 DAYS

BETWEEN

STAGES -

1.4 3.3 2.9 2.5 1.5

.p a” 1.0

:II

y I

I STAGES OF METAMORPHOSIS

FIG. 4. The in uiuo rate of incorporation of 3Hthymidine into the TCA-precipitable fractions (O--O) and the TCA-soluble fractions (x---x) of tadpole tail at various stages of spontaneous metamorphosis. All animals were labeled with 3H-thymidine 60 min before sacrifice. Each point represents the mean of six separate experiments; the vertical bars signify the standard error of the mean. Notation and values in upper portion of figure entitled “Days Between Stages” represents the mean, obtained from 12 tadpoles, of the time required to complete the transition from one stage to the next stage. The transition from stage X to stage XIX normally occurs in 6-12 months.

be to label protein for an extended period with a labeled amino acid, and then follow the rate of increase in the radioactivity of the soluble pool following a cold chase to clear the pool. This method, however, would be complicated by changes in the kinetics of the amino acid pool such as loss of label from the pool to the circulation, or reincorporation of label into protein (which would also make the method dependent upon the rate of protein synthesis). These difficulties could be obviated by the administration of a second isotopic form of the amino acid. After the second isotope becomes distributed throughout the pool the ratio of the two isotopes will be independent of pool kinet-

!l’umouer

during

Metamorphosis

371

ics since the two forms are physiologically indistinguishable. An increase in the ratio of the two isotopes would occur, however, as a result of protein degradation. The rate of protein degradation can thus be determined by following the rate of increase in the isotope ratio with time after allowing a suitable period for equilibration of the second isotope with the pool. Preliminary experiments indicated that equilibration was complete within 30 min after injection of the second isotope. In our experiments tritiated leucine was administered first to prelabel the proteins, and then a pulse label of “C-leucine was given. The rate of increase of the 3H : “C ratio in the free amino acid pool was used to calculate the rate of protein degradation as follows: In vitro the quantity of 3H in the amino acid pool after incubation for time t equals the quantity of 3H at time 0 plus the amount liberated from protein during time t. The same is true for 92 thus

-(Wt = (W, + W3WlJ’l) (‘“Ch (‘“C), + X(“C/Fl) where (3H), and (“C), are the respective amounts of 3H and 14C in the pool at time t; ( 3H) 0 and ( “(2) 0 are the amounts at time 0, X is mg protein degraded, and 3H/[p] and “C/[p] are the specific activities of tritium and 14C in the protein expressed as dpm/mg protein where p] is the protein concentration. If we let R2 = (3H),/ (‘“C), and R, = ( 3H)0/( ‘“C), then we obtain by substitution into the above expression

Rt* = R,(‘“C)o + X3WPl) (‘“C), + x(14c/p]) and solving for X we obtain (R, - R, (‘“C), (WF]l) - Rt(“CIFI) For the in vivo studies we can assume that, since a pulse label of “C was given, no 14C was liberated from the protein. Thirty x=

372

DEVELOPMENTAL

BIOLOGY

minutes were allowed for the “C-leucine to distribute itself throughout the amino acid pool; therefore, the 30-min label is considered to be zero time. The expression for calculating the quantity of protein degraded in vivo is then Rt = Gi1*cC30

+ X3WPI)

(‘“C),, which is equivalent to x = m

+ 0

- R3,) (‘“C),,

3H/Pl The rate of protein degradation which we obtained from this expression for stage XXII was 2.2 x 10m5 mg protein degraded per milligram of tissue per minute. At this rate of protein degradation, approximately 3.7 days would be required to complete the resorption of the tadpole tail. This figure closely approximates the actual number of days between stages XXII and XXIV of spontaneous metamorphosis (Fig. 1) in which tail resorption normally occurs. These experiments have revealed the time sequence of several biochemical changes which occur during tail regression. Between stages XVII and XX there was a progressive decrease in the rate of incorporation of labeled amino acids into protein. An increase in the specific activity of the amino acid pool occurred during this period so that the decrease in protein synthesis is probably more pronounced than is indicated by the decrease in the rate of incorporation. No change in the rate of protein degradation was observed until after stage XX. Such an alteration in the balance between protein synthesis and degradation would be expected to result in a decrease in the quantity of protein per cell. That this actually occurs is indicated by the decline observed in the tadpole tail protein : DNA ratio beginning between stages XIX and XX. The increase in protein and DNA when expressed per gram of tissue is probably the result of dehydration (Lapiere and Gross, 1963). Although a

VOLUME

30, 1973

substantial decrease in the rate of protein synthesis occurs in the tail during metamorphosis, there is considerable evidence that the synthesis of certain specific proteins, notably cathepsins, increases, probably as a result of the activation of macrophages (Weber, 1967). Gross indications of tail resorption, i.e., loss of weight and length, do not appear until some time between stages XX and XXI. It is also during this period that an increase in the rate of protein degradation occurs. The increase in protein degradation which we observed in vivo was confirmed in vitro. Our findings that in vitro protein degradation occurs much more rapidly at pH 3.3 than at pH 6.8 provides strong evidence for the involvement of cathepsins in tail resorption. The decreases observed in the rates of protein and DNA synthesis coincide with the beginning of a rise in the level of circulating endogenous thyroid hormones as measured by protein-bound iodine (Just, 1968). Microscopic examination of tadpole tails at the onset of metamorphic climax (Weber, 1964) reveals changes which are indicative of cell deaih. The morphological changes are followed by the appearance of macrophages and increases in hydrolase activity (Weber, 1967) which coincide with the rise in the rate of protein degradation observed both in vivo and in vitro. Two mechanisms thus appear to be involved in the loss of protein from the tadpole tail during metamorphosis. The first is the depression of the rate of protein synthesis. The intracellular proteases of the tail tissue apparently remain active since no change in the rate of protein degradation was observed until the later stages. This alteration of the normal balance between protein synthesis and degradation, which occurs before the appearance of gross signs of the tail resorption, results in an early loss of protein from the cells and is probably indicative of cell death. The second mechanism apparently

Lrm~,

ATKINSON,

AND FRIEDEN

Protein

involves phagocytosis of cell debris by macrophages and digestion of the remaining protein by proteases contained therein. The precise mechanism by which protein synthesis is depressed and protein degradation (macrophage proliferation) is stimulated as well as the mechanisms by which thyroid hormones influence these processes remain to be elucidated. REFERENCES

ARIAS, I. M., DOYLE, D., and SCHIMKF, R. T. (1969). Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J. Biol.

Chem.

244.3303-3315.

ATKINSON, B. G. (1971). Patterns of macromolecular biosynthesis during amphibian metamorphosis. “Proceedings of 7th Conference on Endocrinology and Metabolism,” pp. 48-82. Univ. of Missouri Press. ATKINSON, B. G., ATKINSON, K. H., JUST, J. J., and FRIEDEN, E. (1972). DNA synthesis in Ranu catesbeiana tabpole liver during spontaneous and triiodothyronine-induced metamorphosis. Deoelop. Biol. 29, 162-175. BURTON, K. (1968). Determination of DNA concentration with diphenylamine. Methods Enzymol. 13B, 163-166. EATON, J. E. (1971). Purine and RNA metabolism during induced metamorphosis in tadpole liver: A possible mechanism of action. Ph.D. Thesis, Florida State Univ., Tallahassee. FOLCH, J., LEES, M., and SLOANE STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. FRIEDEN, E.,

226,497-509.

and JUST, J. J. (1970). Hormonal responses in amphibian metamorphosis. In “Bio-

Turnover

during

Metamorphosis

373

chemical Action of Hormones” (G. Litwack, ed.), Vol. I, pp. l-52, Academic Press, New York. JUST, J. J. (1968). Thyroid hormone and protein concentration in plasma and pericardial fluid of metamorphosing anuran tadpoles. Ph.D. Thesis, Univ. of Iowa, Iowa City, Iowa. LAPIERE, C. M., and GROSS, J. (1963). Animal collagenase and collagen metabolism. In “Mechanisms of Hard Tissue Destruction” (R. Sognaes, ed.), p. 663. Publ. No. 75, Amer. Ass. Advance. Sci., Washington, D.C. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. SCHNEIDER,W. C. (1945). Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid. J. Biol. Chem. 161,293-303. TATA, J. R. (1966). Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture. Deuelop. Biol. 13, 7794. TAYLOR,

A. C., and KOLLROS, J. J. (1946). Stages on the normal development of Ranu pipiens larvae. Anat.

Rec. 94,7-24.

TONOUE, T., and FRIEDEN, E. (1970). The effect of triiodothyronine on leucine incorporation into tail and other tadpole tissues. J. Biol. Chem. 245, 2359-2362.

WEBEP., R. (1964). Ultrastructural changes in regressing tail muscles of Xenopus larvae at metamorphosis. J. Cell Biol. 22,481-487. WEBER, R. (1967). Biochemistry of amphibian metamorphosis. In “Biochemistry of Animal Development” (R. Weber, ed.), Vol. II, pp. 227-301. Academic Press, New York. YEMM, E. W., and COCKING,E. C. (1955). Determination of amino acids with ninhydrin. Analyst 80,209213.